This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Why do defects in molecular machines (AAA+ ATPases) cause disease? These protein machines convert ATP hydrolysis into mechanical work. Both human cells and disease causing pathogens use this work to physically manipulate proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. We do not know how these molecular machines convert ATP hydrolysis into mechanical work. Our research focuses on one subset of AAA+ ATPAses, the bacterial-enhancer-binding proteins (EBPs) which use their ATPase activities to regulate transcription of genes needed for harmful activites (diseases, crop damage) or helpful ones (nitrogen fixation, environmental remediation, hydrogen or other metabolite production). We are addressing the underlying mechanism of AAA+ ATPase function via structure function studies of mutant forms of ATPase. We have combined the functional studies with analysis of a new crystal structure for one of the mutant proteins bound to ATP. The analysis yielded a novel perspective of the conformational changes that occur upon the protomers moving from ATP-bound to ADP-bound. This model predicted unexpected residues would be important for function. We mutated one of the key residues, and used SAXS measurements to study its behavior in the presence of nucletotides. That work was just accepted for publication by Structure (Cell Press).